G protein-coupled receptors muscarinic acetylcholine receptors vasopressin receptors structure-function studies muscarinic receptor knock-out mice yeast expression technolgoy gene targeting disulfide scanning mutagenesis receptor random mutagenesis Summary: (I) One major focus of my section is to understand how muscarinic acetylcholine and other G protein-coupled receptors (GPCRs) function at the molecular level. (II) More recently, we employed gene targeting technology in mice to study the physiological and pathophysiological roles of the individual muscarinic acetylcholine receptor subtypes (M1-M5 mAChRs). (I) STRUCTURE-FUNCTION ANALYSIS OF GPCRs GPCRs form one of the largest protein families found in nature, and estimates are that about 50% of drugs in current clinical use act on specific GPCRs or on GPCR-dependent downstream signaling pathways. To understand how these receptors function at a molecular level, we have used different mAChRs and vasopressin receptors as model systems. To elucidate the structural changes involved in ligand-dependent GPCR activation, we developed a disulfide cross-linking strategy that allows the formation of intramolecular disulfide cross-links between adjacent Cys residues, with the receptor present in its native membrane environment (in situ!) (Ward et al., JBC 277, 2247, 2002). Disulfide cross-linking experiments revealed that agonist activation of the M3 mAChR is associated with striking structural changes on the intracellular surface of the receptor protein (Ward et al., JBC 277, 2247, 2002; Han et al., EMBO J., submitted). We recently demonstrated that the agonist-mediated structural changes occurring on the cytoplasmic receptor surface are accompanied by conformational changes in the immediate vicinity of the ligand binding site located close to the extracellular surface of the transmembrane receptor core (Han et al., manuscript in preparation). To facility structure-function studies, we recently expressed several muscarinic and vasopressin receptor subtypes as well as different G protein alpha subunits in yeast. A great advantage of the yeast expression system is that powerful genetic approaches can be applied to study GPCR structure-function relationships, allowing the screening of large numbers of mutant GPCRs or G proteins in a very efficient manner. To study the structural mechanisms governing M3 receptor activation, we employed an M3 receptor-expressing yeast strain that requires agonist-dependent M3 receptor activation for cell growth. Recently, we used receptor random mutagenesis, followed by yeast genetic screens, to identify mutant M3 mAChRs which are constitutively active and which play key roles in M3 receptor activation (Schmidt et al., JBC 278, 30248, 2003; Schmidt and Wess, unpublished results). Class I GPCRs share about 20 highly conserved amino acids, mutational modification of which frequently leads to a loss of receptor function. We recently expressed several non-functional mutant M3 mAChRs containing inactivating point mutations of such highly conserved residues in yeast. Subsequently, we used receptor random mutagenesis, followed by yeast genetic screens, to isolate second-site mutations that can restore function to these mutant M3 receptors (Bo et al., manuscript in preparation). These results indicate that yeast expression technology, combined with receptor random mutagenesis, provides a powerful novel tool to study the molecular mechanisms governing GPCR function and activation. (II) GENERATION AND ANALYSIS OF MUSCARINIC ACETYLCHOLINE RECEPTOR KNOCKOUT MICE The precise physiological and pathophysiological roles of the individual mAChRs (M1-M5) are not well understood, primarily due to the lack of receptor subtype-selective ligands. To address this issue, we, in collaboration with Chuxia Deng's lab at NIDDK, used gene targeting technology to generate M1-M5 receptor-deficient mice (KO mice). The M1-M5 mAChR KO mice were then subjected to a battery of physiological, pharmacological, behavioral, biochemical, and neurochemical tests. This analysis showed that each of the analyzed mAChR KO lines displayed specific functional deficits, indicating that each mAChR subtype mediates distinct physiological functions (for recent reviews, see Wess, Trends Pharmacol. Sci. 24, 414, 2003; Wess, Annu. Rev. Pharmacol. Toxicol. 44, 423, 2004). Importance of M2 and M5 mAChRs for distinct CNS functions Muscarinic cholinergic mechanisms are known to play a key role in most cognitive processes. We recently demonstrated that M2 mAChR KO mice showed significant deficits in behavioral flexibility, working memory, and hippocampal synaptic plasticity (Fedorova et al., submitted). Since impaired muscarinic cholinergic neurotransmission is associated with Alzheimer's disease and normal aging processes, these findings should be of considerable therapeutic relevance. Phenotypical analysis of M5 mAChR KO mice has revealed that M5 receptors are involved in several important physiological functions (reviewed by Wess, Annu. Rev. Pharmacol. Toxicol. 44, 423, 2004). We recently observed that the rewarding effects of morphine and cocaine and the severity of drug withdrawal symptoms are substantially reduced in M5 mAChR KO mice (Basile et al., PNAS 99, 11452, 2002; Fink-Jensen et al., J. Neurosci. Res. 74, 91, 2003). These findings raise the possibility that centrally active M5 receptor antagonists may become therapeutically useful for the treatment of drug addiction. Roles of M3 and M5 mAChRs in ACh-mediated vasodilation We previously showed that M5 receptors mediate the vasorelaxing effects of ACh on cerebral arteries and arterioles (reviewed by Wess, Annu. Rev. Pharmacol. Toxicol. 44, 423, 2004). Interestingly, we recently demonstrated that the ability of ACh to dilate various peripheral arterial blood vessels is predominantly mediated by M3 mAChRs (Lamping et al., Arterioscler. Thromb. Vasc. Biol. 24, 1253, 2004; Khurana et al., Eur. J. Pharmacol. 493, 127, 2004.). This observation indicates that distinct mAChRs regulate the diameter of cerebral and non-cerebral blood vessels. Roles of M1 and M3 mAChRs in the function of salivary glands ACh-mediated activation of glandular mAChRs is considered the predominant mechanism by which salivary secretion is stimulated physiologically. Studies with M1 and M3 mAChR single and M1/M3 mAChR double KO mice showed that muscarinic agonist-mediated stimulation of stimulation of salivary flow is mediated by a mixture of M1 and M3 receptors (Gautam et al., Mol. Pharmacol. 66, 260, 2004). Since impaired salivary secretion is associated with a number of pathophysiological conditions, these findings should be of considerable therapeutic relevance. Critical role of M3 mAChRs in mediating glucose-dependent insulin release Using isolated pancreatic islets prepared from WT and M3 mAChR KO mice, we recently showed that activation of islet M3 receptors leads to a pronounced potentiation of glucose-dependent insulin release (Duttaroy et al., Diabetes 53, 1714, 2004). This observation suggests that stimulation of _eta-cell M3 receptors may represent a useful approach to boost insulin output in type 2 diabetes. Role of M2 and M3 mAChRs in cardiac and pulmonary function We recently demonstrated that in vivo bradycardia caused by vagal stimulation was abolished in M2 mAChR KO mice (Fisher et al., FASEB J. 18, 711, 2004). In contrast, bronchoconstriction in response to vagal stimulation was selectively abolished in M3 mAChR KO mice. In vitro studies with smooth muscle preparations from peripheral airways showed that muscarinic agonist-induced contractile responses were greatly reduced in preparations from M3 mAChR single KO mice (Struckmann et al., Mol. Pharmacol. 64, 1444, 2003). Since altered cardiac or pulmonary vagal tone is involved in a number of pathophysiological conditions, these results should be of considerable therapeutic relevance.